Mycobacteria In Tissues Research Articles

A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains.

Histopathology is an important method for diagnosing extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after use of acid-fast stain (AFS). This study investigated the mechanism of AFS use and the detrimental effect of histologic processing-in particular, xylene deparaffinization-on AFS and mycobacterial detection. The target of the fluorescent Auramine O (AuO) AFS was investigated using triple staining with DNA- and RNA-specific dyes. The effect of xylene deparaffinization on the acid fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared with a novel, solvent-free projected-hot-air deparaffinization (PHAD). Co-localization of AuO with DNA/RNA stains suggests that intracellular nucleic acids are the true target of AFS, producing highly specific patterns. Xylene reduces mycobacterial fluorescence significantly (P < .0001; moderate effect size, r = 0.33). The PHAD process yielded significantly higher fluorescence than xylene deparaffinization in tissues (P < .0001; large effect size, r = 0.85). Auramine O can be applied for nucleic acid staining of mycobacteria in tissues producing typical beaded patterns. Acid-fast staining depends heavily on the integrity of the mycobacterial cell wall, which xylene appears to damage. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly.

Fungi and mycobacteria in tissue

Fungi and mycobacteria are ubiquitous in our environment. Many organisms colonise skin, airways and other sites. Identifying organisms in tissue, along with the characteristic immune response to the organisms is essential in the diagnosis of fungal and mycobacterial infections. Morphological appearances of organisms are often non-specific, staining patterns, cultures and other methods, including molecular techniques are used to specifically identifying organisms. Host reaction patterns to specific organisms can vary from inactive, to purulent, to granulomatous and evolve over the course of the infection. Aspergillus is an example of fungus that produces a variety of reactions it can be a commensal, colonise a cavity, produce an allergic reaction or invade tissue and produce haemorrhagic necrosis. Tissue reaction to mycobacteria u often located deep in the tissue. The diagnosis of mycobacteria leprae is often dependent on locating the organism in tissue.

Fungi and mycobacteria in tissue

Fungi and mycobacteria are ubiquitous in our environment. Many organisms colonise skin, airways and other sites. Identifying organisms in tissue, along with the characteristic immune response to the organisms is essential in the diagnosis of fungal and mycobacterial infections. Morphological appearances of organisms are often non-specific, staining patterns, cultures and other methods, including molecular techniques are used to specifically identifying organisms. Host reaction patterns to specific organisms can vary from inactive, to purulent, to granulomatous and evolve over the course of the infection. Aspergillus is an example of fungus that produces a variety of reactions it can be a commensal, colonise a cavity, produce an allergic reaction or invade tissue and produce haemorrhagic necrosis. Tissue reaction to Mycobacteria Ulcerans changes over time with anergy, purulent and granulomatous phases, with organisms often located deep in the tissue. The diagnosis of Mycobacteria Leprae is often dependent on locating the organism in tissue .

A simple heat-based alternative method for deparaffinization of histological sections significantly improves acid-fast staining results for Mycobacteria in tissue

Histopathology is the study of how disease alters human and animal tissue and is based on the microscopic examination of stained tissue sections. To maintain tissue integrity, preserving it from degradation, it is initially fixed, primarily with formalin, before being treated with alcohol and organic solvents, allowing the infiltration of paraffin wax. The tissue can then be embedded in a mold and sectioned, usually at a thickness between 3 and 5 μm, before staining with dyes or antibodies to demonstrate specific components.As the paraffin wax is insoluble in water, it is necessary to remove it from the tissue section before applying any aqueous or water-based dye solution, to allow the tissue to successfully interact with the stain. This deparaffinization/hydration step is normally carried out using xylene, an organic solvent, followed by hydration using graded alcohols.However, this use of xylene has been shown to have detrimental effects on acid-fast stains (AFS), such as those employed to demonstrate Mycobacterium, including the causative agent of tuberculosis (TB), as the integrity of the lipid-rich wall present in these bacteria may be compromised using xylene.A simple, novel method, Projected Hot Air Deparaffinization (PHAD) removes the solid paraffin from the tissue section without the use of any solvents, which produces significantly improved staining results using AFS. PHAD relies on the projection of hot air onto the histological section to melt and remove paraffin from the tissue, which can be achieved using a common hairdryer.•PHAD relies on the projection of hot air onto the histological section which can be achieved using a common hairdryer.•The blowing force is such that melted paraffin is removed from the tissue in 20 min.•Subsequent hydration allows for using aqueous histological stains with success, such as the fluorescent auramine O acid-fast-stain.

TRANSPLACENTAL TRANSMISSION OF TUBERCULOSIS INFECTION IN COWS INFECTED WITH MYCOBACTERIUM BOVIS

A culture of M. bovis was isolated from liver and spleen homogenate, from tissues and amniotic fluid of 8- and 6-month-old fetuses of cows with local tuberculous changes in the lymph nodes of the lungs. It serves as confirmation of the transplacental infection transmission. The transfer occurred due to filtering virus-like forms of tuberculosis myco-bacteria, which were transformed into non-acid-fast, partially acid-fast and typical forms of tuberculosis mycobacteria in tissues, which can lead to the emergence of endogenous tuberculosis after birth or create an invisible reservoir of infection, because non-acid-fast forms of mycobacterium do not induce the development of hypersensitivity to tubercu-lin. In addition, the persistence of modified and typical mycobacteria in fetuses can lead to immunological tolerance to their antigens.&#x0D; The results obtained confirm the correctness of the regulations on the isolated rearing of calves born in disad-vantaged conditions, followed by fattening and delivery for slaughter.

A Mycobacterium species for Crohn's disease?

In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Morphological diagnosis of tuberculosis under present-day conditions

The paper presents general statistical data on morbidity and mortality rates of tuberculosis, which show positive trends in recent years, with exception of those of its concurrence with HIV infection. The tasks of the morphological diagnosis of tuberculosis are divided into 4 groups: 1) to refine approaches to detecting mycobacteria in tissues; 2) to optimize the postmortem diagnosis of tuberculosis; 3) to optimize the lifetime differential diagnosis of tuberculosis and to develop methods for predicting its course; 4) to study the pathogenesis of tuberculosis from the standpoint of modern views on an infectious process. The data suggesting that the tissue forms of mycobacteria, the types of inflammatory responses, and the specific features of the pathogenesis of tuberculosis call for further investigations are given. To establish the real role of nontuberculous mycobacteria, to study the likelihood that the patient will be superinfected with other M. tuberculosis genotypes, and to elaborate a uniform (clinical, pathogenetic, and morphological) classification of tuberculosis should be also regarded as the most important tasks in its morphological examination.

The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues

BackgroundNucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA.Principal FindingsRegarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89±0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).ConclusionsOur findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.

Extracellular forms of Mycobacterium bovis BCG in the mucosal lymphatic tissues following oral vaccination.

Oral vaccination with BCG provides protective systemic immunity against pathogenic mycobacterial challenge. In this study, the anatomical distribution of Mycobacterium bovis BCG following oral vaccination was investigated. Replicating bacteria in the Peyer's patches and mesenteric lymph nodes were present as solitary rods or clusters of two to three bacteria, the majority of which were isolated ex vivo as extracellular forms. Only a minority were shown to be associated with typical antigen-presenting cells. Acid-fast staining of mast cell granules in lymphoid tissues revealed a potential pitfall for these analyses and may explain previous reports of acid-fast 'coccoid' forms of mycobacteria in tissues.

Improved detection of mycobacteria species in formalin‐fixed tissue sections

To develop an antibody broadly reactive against mycobacterial species, which will improve detection of mycobacteria in tissue sections by immunohistochemistry (IHC). A sheep antisera was developed by immunization with multiple mycobacteria, and was tested by IHC against a range of mycobacteria in tissues from many species, as well as negative tissue controls and other bacteria. The sheep antiserum, MAS-01, reacted with all 18 mycobacterial species tested, but did not react with uninfected inflammatory tissues. Although MAS-01 cross-reacted with two microbial genera which are related to mycobacteria (Corynebacteria and Proprionibacteria), it did not with Nocardia or Actinomyces. The antibody was more sensitive than the Ziehl-Neelsen stain for detection of tissue mycobacteria, and shortened the time required to identify these infections. The MAS-01 antiserum will facilitate rapid identification of tissue mycobacterial infection by histopathologists.

Immunohistochemical and Taqman real-time PCR detection of mycobacterial infections in fish

Real-time PCR and immunohistochemistry (IHC) assays were developed to detect fish mycobacterial infections at the genus level, based on the RNA polymerase β subunit (rpoB) gene and polyclonal anti-Mycobacterium rabbit serum, respectively. The PCR assay positively identified a number of pathogenic mycobacteria including Mycobacterium abscessus, M. avium ssp. avium, M. bohemicum, M. chelonae ssp. chelonae, M. farcinogenes, M. flavescens, M. fortuitum ssp. fortuitum, M. gastri, M. gordonae, M. immunogenicum, M. malmoense, M. marinum, M. montefiorense, M. phlei, M. phocaicum, M. pseudoshottsii, M. salmoniphilum, M. senegalense, M. shottsii, M. smegmatis, M. szulgi and M. wolinskyi. A detection limit equivalent to 10(2) cfu g(-1) was registered for M. salmoniphilum-infected fish tissue. The IHC precisely localized both free and intracellular mycobacteria in tissues and detected mycobacterial infections down to 10(2) cfu g(-1) tissue. Both assays were found to be more sensitive than Ziehl-Neelsen (ZN) staining, where the detection limit was below 8 × 10(3) cfu g(-1) tissue. Although specificity testing of the real-time PCR against a panel of non-Mycobacterium spp. revealed a degree of cross-reaction against pure DNA extracted from Nocardia seriolae and Rhodococcus erythropolis, no cross-reactions were identified (by either real-time PCR or IHC) on testing of formalin-fixed paraffin-embedded (FFPE) tissues confirmed to be infected with these bacteria. The broad applicability of both assays was confirmed by analysis of FFPE tissues from a range of fish species infected with diverse Mycobacterium spp. The results indicate that both assays, alone or in combination, constitute sensitive tools for initial, rapid diagnosis of mycobacteriosis in fish. This should in turn allow rapid application of more specific studies, i.e. culture based, to identify the specific Mycobacterium sp. involved.

Evaluation of In Situ Methods Used To Detect Mycobacterium avium subsp. paratuberculosis in Samples from Patients with Crohn's Disease

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.

The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection

The aim of this study was to investigate the usefulness of polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection (LCM). The PCR DNA amplification method was used to detect M. tuberculosis in granulomas microdissected from one section stained by haematoxylin and eosin (H&E) from a formalin-fixed paraffin-embedded specimen. The results were compared to those obtained from PCR performed from 10 whole paraffin sections of 5 micro m each, and with the histology, culture and the patient's clinical findings. Forty-nine formalin-fixed and paraffin-embedded samples from 49 patients with a histological suspicion of a mycobacterial infection were investigated. Using culture as the reference method, the sensitivity for the detection of M. tuberculosis was 92% and the specificity was 100% using PCR from microdissected granulomas and were similar to those obtained by using PCR from 10 whole sections. The PCR method of examination of microdissected granulomas from deparaffinised sections is a sensitive, specific and rapid method for the detection of M. tuberculosis in formalin-fixed and paraffin-embedded samples. The method is as sensitive as that using PCR on 10 whole tissue sections, thus making it suitable for small biopsies. However, although these methods reduce the delay in diagnosis, culture remains the gold standard for identification of mycobacteria in tissue. Culture also allows for the testing of antibiotic sensitivity of any isolated species, in this way determining appropriate treatment.

Polyclonal Antibodies Raised Against Bacillus Calmette-Guerin, Mycobacterium Duvalii, and Mycobacterium Paratuberculosis Used to Detect Mycobacteria in Tissue with the Use of Immunohistochemical Techniques

Commercially available polyclonal antibodies raised against strains of mycobacteria were used to detect organisms in tissue sections from 34 cases of tuberculosis, leprosy, and atypical mycobacteria. Thirty-two cases of fungal infections, granulomatous inflammation, and sarcoidosis were used as negative controls. Sections stained with the use of antibodies raised against Bacillus Calmette-Guerin (BCG), Mycobacterium duvalii (MD), and Mycobacterium paratuberculosis (MP) were compared with Kinyoun and Fite-stained tissue sections. In caseating granulomata, clumps of mycobacterial debris, cells, and cell fragments stained. In histiocytic granulomata of mycobacterial infections, histiocyte cytoplasm contained both organisms and debris. The three antibodies showed cross-reactivity against the four groups of mycobacteria tested. Mycobacterial staining using immunoperoxidase was apparent in most cases at low-power (scanning) magnification. Thirty-two of 34 cases of mycobacterial infection, including all 24 Kinyoun-Fite-positive cases, were positive for immunoreactive organisms and debris using anti-MD, anti-BCG, and/or anti-MP. Eight of ten cases of culture-proven mycobacterial infection, in which Kinyoun and Fite stains were negative, had immunoreactive organisms or antigen with anti-BCG, MD, or MP. The antibodies also stained organisms in five cases of sporotrichosis in which the organisms were identified as yeast forms in tissue sections.